Automatic electron microscopy tissue processor method

ABSTRACT

A tissue processor is programmed for fixation, dehydration and clearing of electron microscopy size particles and utilizes a porous receptacle for each particle or group of particles of tissue and a processing chamber adapted to contain a plurality of the receptacles. The chamber is connected to a plurality of containers, some of which are refrigerated, and which contain the various processing solutions. The solutions are individually piped to, and from beneath, the chamber through a remotely controlled valve and manifold arrangement which also connects with a metering pump. This arrangement minimizes fluid contamination and allows each solution to be precisely metered, brought to, retained in and drained from the chamber thus allowing the particles to be bathed in the solutions according to an automatic programmed time sequence. The program may be varied as to number of cycles per solution, as to number of solutions per program, as to time per cycle and as to starting and terminal solutions in the program.

United States atent Kinney et al.

1*June 10, 1975 [54] AUTOMATIC ELECTRON MICROSCOPY 3,400,726 9/1968 DuGrail 134/95 TISSUE PROCESSOR METHOD 3,452,710 7/1969 Hentschel 118/7 3,526,203 9/1970 Kinney et a1 118/7 Inventors: Thomas y; J R 3,550,552 12 1970 Archibald 118/7 Pickett, both of Durham, N.C. 3,576,176 4/1971 Pickett 118/500 3,697,299 10/1972 Pickett 117/3 [73] Asslgnee? Trangle Bmmedlcal Equpmen" 3,771,490 11 1973 Kinney et a1 118/7 Inc., Durham, NC.

[ Notice: The portion of the term of this Primary Examiner-Michael Sofocleous patent subsequent to Nov. 13, 1990, has been disclaimed. [57] ABSTRACT [22] Filed: 1973 A tissue processor is programmed for fixation, dehy- 21 1 N 390 950 dration and clearing of electron microscopy size particles and utilizes a porous receptacle for each particle Related Apphcatlon Data or group of particles of tissue and a processing chaml l Division Of 1972, ber adapted to contain a plurality of the receptacles. 3771,490- The chamber is connected to a plurality of containers, some of which are refrigerated, and which contain the 1 Cl 424/3; various processing solutions. The solutions are individuany to and from beneath the chamber 1 Fleld of Search 7, 8, 5O, 429; through a remotely controlled valve and manifold ar- 134/96, 97, 98, 222/136 144, 144-5, rangement which also connects with a metering pump. 190; 95/93; 7/3; 424/3 This arrangement minimizes fluid contamination and allows each solution to be precisely metered, brought 1 References Clted to, retained in and drained from the chamber thus al- UNITED STATES PATENTS lowing the particles to be bathed in the solutions ac- 2,774,364 12/1956 Brobeil 222/136 cording to an automaticiprogrammed time Sequence. 2 959 151 1 9 0 Ehrlich 113/429 The program may be varied as to number of cycles per 3,092,121 6/1963 Broge 134/97 solution, as to number of solutions per program, as to 3,227,130 l/l966 Weiskopf 118/1 1 ti er cycle and as to starting and terminal solutions Suzuki et all in the program 3,350,220 10/1967 lsreeli 117/113 3,392,780 6/1968 Brown 118/429 3 Claims, 11 Drawing Figures lll' PATENTEDJUH 10 I975 3889314 sum CYLES QOWHW' WM JIME- HOLD/PASS illiuilfllimiilil E TIME a: r a a 150 I 155 140 4 6 7 Hi SOLUTION INDICATOR T'MER 1 2 3 4 5 6 7 8 9 10 o 3 HQDUUUUUUUUD 14 POWER 2 9 '11 132 I 15o? 1% i ON 1 10 ll START B C .RELEASE 5 11 OFF SELECTOR 11 A99 {I 10 6 9 H] VOLUME 181 i M 98 95 97 FIG. 5

L. SOL. SOL. SOL. SOL. SOL, SOL. 4 5 6 7 8 9 1O 40! PROP. RAO. ALC ALC. ALC. ALC. ALC. ALC.

POROU S BODY ORGANIC SOLUTION AQUEOUS AND ORGANIC SOLUTION FLOW SPECIMEN NUMBER PATENTEDJUHIOIM 3.8891014 sum 3 FIG. 7

TO WASTE FULL l N DICATOR PATENTED JUH 1 0 I975 SHEET PATENTED JUH 1 0 I975 SHEET AUTOMATIC ELECTRON MICROSCOPYTISSUE PROCESSOR METHOD CROSS-REFERENCE TO RELATED APPLICATIONS This application is a division of copending application, Ser. No. 228,115, filed Febv 22, 1972, entitled Automatic Tissue Processor, now U.S. Pat. No. 3,771,490.

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates broadly to methods for processing pieces of tissue but particularly to methods for processing electron microscopy size tissue.

2. Description of the Prior Art This invention constitutes an improvement upon the subject matter of our prior U.S. Pat. No. 3,526,203, upon the subject matter of U.S. Pat. No. 3,697,299 and upon the methods related thereto. Since the general history of the prior art is dealt with in some detail in prior U.S. Pat. No. 3,526,203 reference is made to U.S. Pat. No. 3,526,203 for the prior art background of the present invention. Reference is also made to prior U.S. Pat. No. 3,536,040 directed to a selectively permeable receptacle and to U.S. Pat. No. 3,576,176 directed to a tissue capsule rack which forms part of the prior art of specific interest and useful in practicing the present invention. In summary, the subject U.S. Pat. No. 3,526,203 discloses a processor for processing electron microscopy size tissue, as well as larger tissue, in a permeable receptacle of the type disclosed in U.S. Pat. No. 3,536,040 with the receptacle being held in a rack as shown in U.S. Pat. No. 3,576,176. Our own prior art is believed to be the closest known prior art. Of particular interest to the present invention is that the processor of our prior patent disclosed a system and a method based on such system of connecting the processing chamber with the processing solution containers by means of pipes which connected to the top of the processing chamber and depended on gravity feed and further depended on sensing the level of the solution in the chamber as a means of stopping flow and measuring the volume of a particular solution. After extensive experimental use it was found that the processor and method of our prior patent inherently introduced substantial opportunity for the solutions to mix in the various pipes between the chamber and the containers. Also, after experimental use with various types of solutions, particularly osmic acid, and time programs the prior processor and the related method was found to introduce a problem in maintaining accurate level control for the various solutions. Certain solutions when passed through relatively long lines tended to precipitate in the lines particularly when the solution was held at or above room temperature and no provision was made in the prior processor or in the method of using it for maintaining selected solutions at temperatures below room temperature.

Of particular interest is the fact that while our prior patent taught the general concept of automatic operation with a program switch the processor and method of the prior patent did not disclose means for conveniently changing the numbers of cycles for a particular solution. That is, in hand processing it is sometimes the 5 practice to hold the tissue in a solution, then drainthe solution, then hold the tissue again in the same solution for another time period. The processor and method of our prior patent did not provide a means for duplicating in a machine operation this aspect of hand processing. While the prior patent processor and method contemplated changing an entire program in order to obtain such a variation in numbers of cycles per solution the processor of our prior patent did not not offer the possibility of making such a change even while a particular program was in progress. Furthermore, the program capability of the processor and method of our prior patent was to some extent limited in being able to vary the amount of time devoted to a particular solution being in the processing chamber. Thus, with the processor and processing method of our prior patent if it was desired to change the base time during which a particular solution was held in the processing chamber from, say, twelve minutes to twenty-four minutes it was necessary to change the entire program for the entire run of solutions. Also, the prior processor and method did not exhibit the capability of being able to pass certain solutions in a program or to terminate a particular program, at least temporarily, with a particular solution. In hand processing of electron microscopy tissue it is sometimes desirable to, say, stain a tissue after processing solutions 1, 2 and 3 and to, say, bypass solution 4 with a certain tissue.

From the foregoing it can thus be seen that while the processor and method of our prior patent constituted an important advance in the art and so far as we know constituted the first significant step toward a practical automatic processor and method for processing electron microscopy size tissue long and extensive experimental use with that processor have revealed a number of needed improvements in terms of both apparatus and method and such improvement in method constitutes the object of the present invention.

SUMMARY OF THE INVENTION The invention is generally directed to a method for processing electron microscopy size tissue and constitutes an improvement over the method called for in our prior U.S. Pat. No. 3,526,203. Reference to our prior patent is made for an understanding of the general operation involved with an apparatus of this kind. Of specific interest to the present method invention, the apparatus used in practicing the method embodies a stationary processing chamber having a bottom port connected directly to a manifold which is positioned below the chamber. The manifold is also provided with a plurality of inlets connected through pipes to each of the respective processing solutions and with an outlet for waste discharge. Each inlet on the manifold mounts a remotely solenoid controlled valve so that the solutions from each solution container can be effectively isolated from each other at points immediately adjacent the manifold which minimizes the opportunity for, say, so lution l to mix with solution 3 and also minimizes the amount of necessary waste solution. The chamber port communicates with a solenoid control valve immediately adjacent the manifold as does the waste outlet. A metering pump also connects to the manifold and incorporates a metering piston driven by a reversible motor so that the piston can be withdrawn to pull in a given amount of solution and can be moved in the opposite direction to force the solution into the processing chamber through the mentioned bottom port. The

manifold thus provides a passage for a selected solution to be withdrawn into the pump and to then be forced into the processing chamber. During withdrawal all valves except the selected solution valve are closed. On the return stroke only the processing chamber valve is opened and during dumping only the waste valve is opened to allow the expended solution to drain by gravity. Means are provided whereby the amount of pump cylinder movement can be precisely regulated to vary the amount of solution withdrawn so as to eliminate the need for level sensing and control as in the processor of our prior patent. The various solenoid valves are also coordinated by program switch means such that all the respective solutions may be metered, pumped up to the processing chamber and then allowed to drain by gravity to waste with minimal mixing of one solution with another except in the collective waste.

For greater variety of programming, the invention also adopts the concept of establishing a so-called base time of, say, l minutes and incorporating means whereby a particular solution may be held during a particular cycle for one, two or three periods of base time. Thus, merely by moving a switching arrangement a solution can be held for, say, 15, 30 or 45 minutes. This can be further varied so that, during a particular program, solution I stays for, say, minutes, solution 2 for 45 minutes and solution 3 for 45 minutes in that sequence. The program can be changed while in progress or from program to program merely by reorienting a set of switch positions. Thus, a medical research investigator is offered a wide range of processing techniques for comparing one mode of processing the same tissue or different tissue particles with another mode of processing. The processor of the invention also mounts a switching arrangement and the method of use is adapted to allow any particular solution to be bypassed in a program or for the processing to stop, at least temporarily, when such solution is reached. An improved ventilation system has also been provided which during use of the processing method allows fumes in the work area and particularly around the processing chamber to be withdrawn as rapidly as formed.

In summarizing the present invention in method terms, it can be said that the method of the invention minimizes intermixing of solutions, increases accuracy of measuring and provides greater variety in programming. Other improvements of an important but lesser nature will also be noted as the description proceeds.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view of an apparatus for processing electron microscopy tissue according to the invention method.

FIG. 2 is a right elevation view of the apparatus.

FIG. 3 is a rear elevation view of the apparatus.

FIG. 4 is a left elevation view of the apparatus with various compartment doors open.

FIG. 5 is an enlarged front elevation view of the main control panel and also showing the auxiliary control panel in an open position.

FIG. 6 is a front elevation view of the solution containers with the container compartment doors being shown in an open position.

FIG. 7 is a perspective somewhat schematic view of the manifold, metering pump, processing chamber and waste tank arrangement.

FIG. 8 1s a sectional and somewhat schematic elevation view of the chamber. valve and metering pump arrangement.

FIG. 9 is a plan, sectional and somewhat schematic view of the manifold, passages. valves and metering pump.

FIG. 10 is a schematic diagram of the control system.

FIG. 11 is a perspective view ofa prior art porous receptacle used with the invention method.

DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention is directed to a processing method for processing tissue particles held in a porous receptacle 25 of the type shown in FIG. 11 and more fully disclosed in related US. Pat. No. 3,536,040. A typical receptacle of this type is porous at least in some portion, whether in the body 20 or the end caps 21, to all the solutions. It may be roughly /2 inch in length and /2 inch in diameter and the pore size in the range of about 40 to 200 microns. A substantial number of such receptacles may be processed simultaneously and according to a time schedule for the various processing solutions as later discussed.

The processor used in practicing the method invention includes a cabinet 29 having pivoted front doors 30, 31, a pivoted rear door 32 and a pivoted top door 33. Doors 30, 31 cover, respectively, a refrigerated compartment 40 and a non-refrigerated compartment 41 having a dividing partition 42 and a back wall parti tion 43. Suitable heat insulation 45 is mounted on door 31 and surrounding compartment 40 in order to retain the reduced temperature obtained by suitable refrigeration mechanism generally indicated at 46. Ten solution containers 50-59 are shown by way of example and are labeled respectively: solution 1, 4% glutaraldehyde; solution 2, buffer; solution 3, 1% osmium tetroxide; solution 4, 50% alcohol; solution 5, alcohol; solution 6, 80% alcohol; solution 7, 95% alcohol; solution 8, absolute alcohol; solution 9, propylene oxide; solution 10, propylene oxide-Epon 50-50. The first three mentioned solutions have been found to require refrigeration for best processing results and it is for this reason that compartment 40 is refrigerated. Each solution container is provided with a suitable quick-disconnect 70, FIG. 6, and an air inlet 71 to allow the solution to be withdrawn through a connecting tube 72. The respective connecting tubes 72 are each connected to a respective solenoid valve 73 which in turn are mounted on a manifold structure 75. Structure 75, as shown in FIG. 9, provides a series of short passages for communicating all of the various solution, chamber, waste and pump inlets and ports. Like other components exposed to the solutions, structure 75 must be of a material inert to all the solutions and stainless steel and Teflon plastic have been employed. The ten tubes 72 leading from the ten solution containers 50-59 are connected to ten respective solenoid valves 73. Manifold 75 also mounts above the manifold a solenoid valve having a connecting pipe 81 connected to the processing chamber 90 and a pipe 82 connected to the manifold 75. Below the manifold there is mounted a further solenoid valve 85 having a pipe 86 connecting to the manifold 75 and a pipe 87 connecting to a waste tank 88. The exposed portions of the valves must be inert and are preferably stainless steel with Teflon inserts though other materials may be employed if suitably inert to the solutions.

Manifold 75 is also connected through a pipe 100 with a pump metering chamber generally indicated at 101. All parts of the metering pump exposed to the solution should be relatively inert. Teflon plastic and stainless steel have been employed for this purpose. Chamber 101 is formed in a piston operated metering pump which is designated 99 and in this case includes a reversible electric motor 105, FIG. 7, connected to drive an internally threaded tube member 106 which in turn receives a threaded piston rod 107 such that when the armature of motor 105 rotates in one direction piston rod 107 is moved forwardly to move piston 110 toward the cylinder port 111 and when the motor armature turns in the opposite direction piston 110 is moved away from port 111. Piston rod 107 mounts a pin 112 which moves back and forth in a slot 113 located in bracket 114. Four micro-switches 115, 116, 117 and 118 are mounted in the path of retracting movement of pin 112 so as to strike pin 112 and be actuated. Switches 115, 116, 117 and 118 are arranged in the circuitry so that one of the switches can be selected to control the amount of movement of pin 112 during retraction by deenergizing motor 105 and by this means very precisely control the amount of solution metered by pump 99 and indicated by levels A, B, C and D. It should be understood that appropriate circuitry commands motor 105 to turn to advance piston 110 when a solution is to be metered, other circuitry commands piston 110 to retract, other circuitry selectively con trolled by micro-switches 116-118 command motor 105 to stop and other circuitry commands motor 105 to turn so as to advance piston 110 to pump out the me tered solution. Annular piston edges 109 are formed to assist sealing.

The processing chamber 90 of the present invention is generally like but differs in an important respect from that shown in prior U.S. Pat. Nos. 3,526,203 and 3,576,176. Specifically, the solutions, instead of being admitted at the top through plural inlets, are both admitted and discharged through a single port 91 in the bottom of chamber 90. Level sensing is thus avoided by means of the described metering pump. Chamber 90 has a tapered bottom which assists in dumping solutions and also allows a relatively small number of capsules to be processed. To describe the processing chamber 90 in more detail it will be seen that the re movable cap 120 is provided with a clear glass window 121. Chamber 90 is physically located within cabinet 29 near refrigerated compartment 40 which minimizes solution precipitation, particularly that of osmium tetroxide. Cap 120 is positioned slightly above the horizontal cabinet wall 123 which provides a relatively large unobstructed work surface to the side of processing chamber 90. The chamber should be inert to all solutions and has been made of Teflon plastic, Stainless steel and other materials may be used. As shown in FIG. 8, a small vent hole 124 is provided on the upper inner wall of the processing chamber which allows the solutions to drain and also allows the fumes from solutions to be exhausted through an outside hole 125 by an exhaust fan 130 connected through a flexible conduit 131 to a suitable hood, not shown. That is, any fumes escaping through holes 124, 125 are drawn into the interior of cabinet 29 and then exhausted by fan 130. Also, when cap 120 is removed any fumes escaping from chamber 90 are drawn into apertures 126 in top wall 123 as shown in dotted lines in FIG. 1. The

fumes are then drawn inside cabinet 29 and exhausted by fan 130. During processing the capsules are secured in a tissue capsule rack of the type described in U.S. Pat. No. 3,576,176, indicated at 92, FIG. 8.

The processing of tissue particles according to the method invention has been described as generally involving placing the tissue particles in the receptacles; placing the receptacles in the receptacle rack, placing the rack in the chamber and then drawing the solutions into the processing chamber in some predetermined time sequence. In this regard the present invention eliminates level sensing in chamber and allows the amount of each fluid drawn into the processing chamber to be precisely metered according to the number of receptacles being processed. Thus, the panel board in FIG. 5 and 10 will be seen to have a volume control switch 132 marked A, B, C and D which correspond in one embodiment to respective loads of l receptacle, (10 ml.), 2 to 4 receptacles (25 ml.), 5 to 28 receptacles (50 ml.) and 29 to 48 receptacles ml.). Switch positions A, B, C and D effectively select a corresponding mirco-switch from the group of pump microswitches 115, 116, 117 and 118 and thereby control the level of fluid used of a particular solution. Additional program circuitry, not shown, commands the pump to otherwise start and stop as required.

The circuitry with which the method is practiced also includes what is called a cycle control which is shown at 135 in FIGS. 5 and 10 and which allows either 1, 2 or 3 solution changes of each stock solution. That is, the group of ten, three position switches shown in FIGS. 5 and 10 at 135 are suitably connected into the illustrated program control circuitry so that each solution can be controlled during a particular program as to the number of changes 1, 2 or 3 of that solution. Thus, the cycle switch for solution 1 may be set to call for solution 1 to have one change, the cycle switch for solution 2 may be set to call for solution 2 to have two changes, the cycle switch for solution 3 may be set to call for solution 3 to have one change and so forth, as shown in FIG. 10. Furthermore, cycle switches may be set one way for one program and immediately after that program is terminated the switches may be moved to a different array for the next program. In this manner, a versatility in method not heretofore achieved is provided. Those skilled in the art will, of course, understand that the cycle switches by their settings control whatever program circuitry is employed. FIG. 10 is intended to simply illustrate one such configuration based on using a stepping switch and standard logic type circuits.

In another aspect of the programming, the present method invention also provides for use of a so-called time control. The time control operates on the basis that each program is made up of some multiple of a standard time, e.g., 15 minutes. A switch 150, shown in FIGS. 5 and 10, is a multiple position timing switch and each position corresponds to a portion of the base time period of 15 minutes. That is, switch can be set for any time from O to 15 minutes. In addition to switch 150 the time control also includes for each of the ten solutions a multi-position time switch indicated at 155, which can be .moved to a position to indicate either one, two, three, four, five, six etc., base units of time. Thus, if the timing switch 150 is set on ten minutes and the particular time switch for solution 1 is set on the 2 position this would mean that solution 1 would stay in processing chamber 90 for 20 minutes (two times the base ten unit) and then run out. That is. each cycle with solution 1 would be for 20 minutes. If the corresponding cycle control switch 135 for solution 1 were set on its 1 position the exchange of the solution 1 with the particles would only take place once but if, for example, the cycle control switch 135 were set on, say, its 2 position the exchange would be repeated for another 20 minute period. The base time control circuitry as well as the cycle control circuitry is, of course, connected to suitable programming circuitry as generally illustrated in FIG. 10. Thus, any solution can be regulated independently of all other solutions as to the number of cycles and the time per cycle merely by positioning the switches I35, 150 and 155.

The circuitry with which the method invention is practiced also incorporates a set of ten, three position, hold-pass switches 170 having positions indicated by N for normal, P for pass and S for stop. Switches 170 are suitably connected into the program circuitry as indicated in FIG. 10. As background information it may be noted that tissue processing of electron microscopy size tissue is subject at times to many collateral operations for experimental purposes or to get certain results supplementary to those achieved by routine processing. That is, in some cases a program can be set up which simply calls for the tissue particles to be processed step by step through all the solutions. In other cases however the research investigator may, for example, want to bypass certain solutions or he may want to, at least temporarily, terminate the program after a particular solution. When the particular hold-pass switch 170 is set on N for normal the solution is used in a normal way and the switch position has no effect on the program. However, when a selected hold-pass switch 170 is set on P this switch setting directs the program circuitry to pass the respective solution in the program and not use it. Thus, if the hold-pass switch 170 is set on P for solution 3, this solution would not be used at all. Also, if the particular hold-pass switch 170 is set on S the associated program circuitry is so arranged as to call for the program to shut down immediately after such solution has been dumped. Thus, if the operator technician is directed to stain all the particles in a special stain solution between, say, solutions 6 and 7 the technician may set the hold-pass switch 170 corresponding to solution 6 to the S position and the program will temporarily stop after solution 6 has been dumped to allow this collateral staining operation.

Considering further the circuitry and control panel with which the method is practiced as shown in FIGS. and 10, there is also incorporated a selector switch 160 having ten positions, corresponding to the ten solutions, and which allows the operator of the processor to select as a starting point in any program any of the ten respective solutions. Thus, a particular tissue may require special processing, in only, say, solutions 7, 8, 9 and 10 and this can be immediately obtained dependent only on a proper initial setting switch 160.

To complete the general explanation of the circuit with which the method invention is practiced it will be understood that given the concept of providing switch means to vary the base time, the cycles per solution, the particular solution starting point, the time per cycle per solution and processing chamber level that many and varied logic, stepping switches and other program and timing arrangements other than that shown generally and substantially in FIG. 10 may be employed in conjunction with such switch means. That is, the ten cycle switches I35, the ten hold-pass switches 170, the ten time switches 155, the selector switch 160, the volume switch 132 and the base time switch 150 may be used to control a variety of program circuits suitable to the invention and well known in the art of programming. The circuitry also includes a start switch 180 whose purpose, and circuit arrangement, is to start a selected program once the various program selected switches have been positioned as required for such program. In addition there is provided a release switch 181 and a so-called clean switch 182. The purpose of release switch 181 is to provide a means to operate the dump valve so that in the event of a malfunction or for other reasons it becomes necessary to immediately drain chamber this can be done by actuating release switch 181. Switch 181 is wired accordingly to override all other program commands. The clean switch 182 serves another useful purpose and is wired in the circuitry so as to command only certain solution control valves 73 to operate in some predetermined sequence designed to flush or clean out the chamber 90, manifold 75 and the various passages at the end of one program and before another program starts. Thus, clean switch 182 effectively commands a subsidiary program intended for cleaning the system and not for processing as such. In one embodiment clean switch 182 and the associated circuitry are programmed to dump fluid from chambr 90, thn to rinse twice with propylene oxide, solution 9, then once with absolute alcohol, solution 8, then to dump and dry. Indicator lamps 95, 96 and 97 indicate as shown in FIG. 10. Lamp 94 indicates a waste tank full condition.

It may be mentioned that in an alternate method a manifold arrangement has been employed experimentally with level sensing as distinct from positive metering. In the alternate arrangement the manifold allowed the solutions to flow by gravity and entry was made through a single port located at the top of the processing chamber, however, this was found not to offer the advantages of the present invention. The illustrated manifold of the invention has proven particularly advantageous in providing a means for shortening flow passages and for minimizing intermixing of solutions. It also provides a means for mounting the solution control valves, the dump valve and the processing chamber valve as well as the metering pump. The use ofa single solution entry and discharge port in the processing chamber reduces possible points of contamination from ten to one. Thus, while the method and apparatus of our own prior art worked and provided a highly significant advance in the art it can be seen that the present manifold arrangement facilitates use of a method which has further advanced the art and in novel and significant respects.

Agitation and vibration of the solutions during processing to increase solution penetration through the receptacle pores has been tried but is generally not deemed necessary. Also, the required agitation or vibration apparatus has not been found to be justified by the results obtained. However, it is, of course, recognized that vibration or agitation may be employed and may in the future, as automatic tissue processing knowledge becomes more fully developed, prove justifiable in certain tissue techniques. Thus, a vibrator V, FIG. 8, may be employed to vibrate chamber 90 or a motor M, FIG. 8, may be employed to rotate rack 92 during processing somewhat like an electric ice cream freezer dasher to obtain solution agitation.

While not shown. the control panel and circuitry used in practicing the method may include, depending on the specific program circuitry employed. a set switch to set the program logic circuitry once the external control switches have been selectively manually positioned and a reset switch to restore such logic circuitry to a particular program state. Such set and reset switches have been employed in one experimental embodiment and a starting sequence used which used the solution indicator lamps shown in FIG. to indicate to the operator that a selected program has been set and is ready to start on a particular solution indicated by the associated solution indicator lamp, in solution indicator lamp bank 140.

What is deemed most significant and most important to providing a reliable day-to-day automatic electron microscopy processing method is the achievement in this invention of a practical method of measuring and of minimizing fluid contamination. Also, of special importance to the present invention has been the achievement and use of a manual switch selector system that readily adapats to any of many well known forms of program circuitry to allow selection of base time, number of solution cycles, starting solutions, positive level measuring, particular solutions for particular processing and cleaning programs, and alternate terminal solutions for particular programs. The research investigator and histology technician is thus provided with a new apparatus and method for both duplicating hand processing procedures and, more importantly, for allowing completely new processing techniques not heretofore known in the art.

While primarily directed to electron microscopy size tissue, it is recognized that much of the fluid exchange and timing apparatus of the invention lends itself to automatic slide tissue staining as well as to light microscopy processing directed to processing 3 to 5 millimeter thick piece of tissue. For staining slides the processing chamber of the invention may be in the form of a rectangular well and used in conjunction with a conventional staining slide tray adapted to hold, for example, 6O 3 X 2 slides. The staining fluids would be recirculated rather than dumped but would be periodically changed depending on the amount of use. The solenoid valves and pump operation could be controlled accordingly and such staining could be performed at room temperature. For light microscopy work larger solution containers should be employed and all lines and pump surfaces handling paraffin should be maintained at a temperature high enough to keep the paraffin in a fluid state. The tissue receptacles for such light microscopy processing could be of the type shown in US. Pat. No. 3,41 1,185 and a plurality of such receptacles could be stacked in a perforated basket for moving into and out of the processing chamber. The processing chamber could be the same rectangular well form used for staining thus adding to the versatility. As with staining the fluids would normally be recirculated and selected fluids periodically changed depending on the amount of use.

What is claimed is:

l. The method of processing in a closed, automatic processor wherein a plurality of individual electron microscopy size particles of the character of having at least one dimension no greater than one millimeter are separately contained while being bathed simultaneously according to a predetermined program for varying lengths of time in successive selected aqueous and non-aqueous electron microscopy tissue processing solutions drawn from separate containers thereof so as to prepare the particles preparatory to embedding. comprising the steps of:

a. separating the particles to be processed into groups;

b. installing and physically isolating each group in a respective uniform tissue receptacle which is porous to the extent of having for each solution at least some porous portion permeable thereto;

c. placing a plurality of such receptacles in an open top processing chamber having a removable top cover, an entry-discharge port in the bottom thereof and a vent hole proximate the top cover and replacing the cover;

d. in a predetermined time sequence and for each solution cycle:

1. withdrawing from one of said solution containers into a controllable metering means located externally of said processing chamber a metered amount of said solution used in such cycle;

2. connecting said external metering means holding said metered solution to said port and with said port open forcing said metered amount of said solution from said external metering means into said chamber through said port and then closing said port;

3. retaining the said metered amount of solution for a predetermined amount of time in said chamber;

4. opening said port and while blocking return of the used solution to said external metering means allowing the used solution to drain from said chamber through said port; and

5. during each such sequence holding the receptacles successively submerged in each respective solution and in a depth of solution determined solely by said metered amount thereby allowing each of the selected solutions to be exchanged with each group of particles by penetration and drainage through said pores while maintaining said system closed, a fixed amount of solution in said chamber and each said group physically isolated from the other.

2.. The method of claim 1 wherein said metering operation is temporarily terminated after a selected solution and prior to cycling all said solutions.

3. The method of claim 1 wherein one of said solutions is cycled more than once before proceeding to the next selected solution, wherein one of said solutions is bypassed in the program, wherein said metering sequence is temporarily terminated after a selected solution and prior to cycling all said solutions, wherein the amount of time said solutions are held in said chamber is non-uniform, and maintaining selected said solutions at a temperature below ambient. 

1. The method of processing in a closed, automatic processor wherein a plurality of individual electron microscopy size particles of the character of having at least one dimension no greater than one millimeter are separately contained while being bathed simultaneously according to a predetermined program for varying lengths of time in successive selected aqueous and nonaqueous electron microscopy tissue processing solutions drawn from separate containers thereof so as to prepare the particles preparatory to embedding, comprising the steps of: a. separating the particles to be processed into groups; b. installing and physically isolating each group in a respective uniform tissue receptacle which is porous to the extent of having for each solution at least some porous portion permeable thereto; c. placing a plurality of such receptacles in an open top processing chamber having a removable top cover, an entrydischarge port in the bottom thereof and a vent hole proximate the top cover and replacing the cover; d. in a predetermined time sequence and for each solution cycle:
 1. withdrawing from one of said solution containers into a controllable metering means located externally of said processing chamber a metered amount of said solution used in such cycle;
 2. connecting said external metering means holding said metered solution to said port and with said port open forcing said metered amount of said solution from said external metering means into said chamber through said port and then closing said port;
 3. retaining the said metered amount of solution for a predetermined amount of time in said chamber;
 4. opening said port and while blocking return of the used solution to said external metering means allowing the used solution to drain from said chamber through said port; and
 5. during each such sequence holding the receptacles successively submerged in each respective solution and in a depth of solution determined solely by said metered amount thereby allowing each of the selected solutions to be exchanged with each group of particles by penetration and drainage through said pores while maintaining said system closed, a fixed amount of solution in said chamber and each said group physically isolated from the other.
 2. The method of claim 1 wherein said metering operation is temporarily terminated after a selected solution and prior to cycling all saiD solutions.
 2. connecting said external metering means holding said metered solution to said port and with said port open forcing said metered amount of said solution from said external metering means into said chamber through said port and then closing said port;
 3. retaining the said metered amount of solution for a predetermined amount of time in said chamber;
 3. The method of claim 1 wherein one of said solutions is cycled more than once before proceeding to the next selected solution, wherein one of said solutions is bypassed in the program, wherein said metering sequence is temporarily terminated after a selected solution and prior to cycling all said solutions, wherein the amount of time said solutions are held in said chamber is non-uniform, and maintaining selected said solutions at a temperature below ambient.
 4. opening said port and while blocking return of the used solution to said external metering means allowing the used solution to drain from said chamber through said port; and
 5. during each such sequence holding the receptacles successively submerged in each respective solution and in a depth of solution determined solely by said metered amount thereby allowing each of the selected solutions to be exchanged with each group of particles by penetration and drainage through said pores while maintaining said system closed, a fixed amount of solution in said chamber and each said group physically isolated from the other. 